Difference between revisions of "Part:BBa K861020"

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The first step in the beta-oxidation pathway involves the conversion of acyl-CoA into enoyl-CoA. This process is catalyzed by acyl-CoA dehydrogenase FadE. Its action results in the introduction of a trans double-bond between C2 and C3 of the acyl-CoA thioester substrate.
 
The first step in the beta-oxidation pathway involves the conversion of acyl-CoA into enoyl-CoA. This process is catalyzed by acyl-CoA dehydrogenase FadE. Its action results in the introduction of a trans double-bond between C2 and C3 of the acyl-CoA thioester substrate.
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===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K861020 SequenceAndFeatures</partinfo>
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===Functional Parameters===
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<partinfo>BBa_K861020 parameters</partinfo>
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== Characterized by BNU-China 2019 ==
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In order to have our engineered microbe consume the extra in-taken fat, we overexpress fadE gene derived from E. coli K-12 DH5alpha genome in our engineered intestinal microbe to catalyze dehydrogenation process from fatty acyl-CoA to fatty enoyl-CoA in fatty acid beta oxidation.
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[[Image:2019_BNU-China_jpgBBa_K861020_pic1.jpg | border | center | 400px]]<br>
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Considering that sodium oleate has a generally steady and relatively high content in most kinds of fat, we select it to test relative general consumption of higher fatty acids.
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We take E. coli introduced with a vector with the same backbone as control group. Compared to it, the experimental group shows an increase in fatty acids consumption upon induction. As is shown in Fig. 1, the experimental group consumes nearly twice as much sodium oleate as the control group within 2 and 4 hours, indicating enhancement of β-oxidation consume an extra amount of higher fatty acids is achieved by overexpressing fadE gene.
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[[Image:2019_BNU-China_jpgBBa_K861020_pic2.jpg | border | center | 400px]]<br>
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<div class = "center">Figure 1 Consumption of sodium oleate</div>
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<font size="4"><b>Experimental approach</b></font>
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1.Transform the plasmids into E. coli DH5α competent cells.<br>
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2.A strain containing a vector with same backbone is used as control. Experimental groups and control groups are cultured in LB-ampicillin (50 ng/µl) medium overnight before being diluted with equal amount of LB-ampicillin (50 ng/µl) medium containing 400 mM sodium oleate, making the final concentration of oleate 200 mM.<br>
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3.Both groups are induced with 5 mM IPTG and sampled at 0 hr, 2 hr and 4 hr. Centrifuge samples and take the supernatant.<br>
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4.Measure the fatty acids concentration through enzyme linked immunosorbent assay (Shuangying FFA ELISA kit).<br>
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5.Calculate and compare the sodium oleate consumption of experimental group and control group.<br>
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6.Three repicas are tested in each group.<br>
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 10:16, 9 October 2019

FadE ,acyl-CoA dehydrogenase

The first step in the beta-oxidation pathway involves the conversion of acyl-CoA into enoyl-CoA. This process is catalyzed by acyl-CoA dehydrogenase FadE. Its action results in the introduction of a trans double-bond between C2 and C3 of the acyl-CoA thioester substrate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 584
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 367
    Illegal SapI.rc site found at 1240
    Illegal SapI.rc site found at 2383



Characterized by BNU-China 2019

In order to have our engineered microbe consume the extra in-taken fat, we overexpress fadE gene derived from E. coli K-12 DH5alpha genome in our engineered intestinal microbe to catalyze dehydrogenation process from fatty acyl-CoA to fatty enoyl-CoA in fatty acid beta oxidation.

2019 BNU-China jpgBBa K861020 pic1.jpg

Considering that sodium oleate has a generally steady and relatively high content in most kinds of fat, we select it to test relative general consumption of higher fatty acids. We take E. coli introduced with a vector with the same backbone as control group. Compared to it, the experimental group shows an increase in fatty acids consumption upon induction. As is shown in Fig. 1, the experimental group consumes nearly twice as much sodium oleate as the control group within 2 and 4 hours, indicating enhancement of β-oxidation consume an extra amount of higher fatty acids is achieved by overexpressing fadE gene.

2019 BNU-China jpgBBa K861020 pic2.jpg

Figure 1 Consumption of sodium oleate

Experimental approach

1.Transform the plasmids into E. coli DH5α competent cells.
2.A strain containing a vector with same backbone is used as control. Experimental groups and control groups are cultured in LB-ampicillin (50 ng/µl) medium overnight before being diluted with equal amount of LB-ampicillin (50 ng/µl) medium containing 400 mM sodium oleate, making the final concentration of oleate 200 mM.
3.Both groups are induced with 5 mM IPTG and sampled at 0 hr, 2 hr and 4 hr. Centrifuge samples and take the supernatant.
4.Measure the fatty acids concentration through enzyme linked immunosorbent assay (Shuangying FFA ELISA kit).
5.Calculate and compare the sodium oleate consumption of experimental group and control group.
6.Three repicas are tested in each group.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 584
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 367
    Illegal SapI.rc site found at 1240
    Illegal SapI.rc site found at 2383